BACKGROUND: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. METHODS: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. RESULTS: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. CONCLUSIONS: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Hospital Veterinari Canis Girona Spain

ISGlobal Hospital Clínic Universitat de Barcelona Barcelona Spain

Secció de Parasitologia Departament de Biologia Sanitat i Medi Ambient Facultat de Farmàcia i Ciències de l'Alimentació Universitat de Barcelona Barcelona Spain

See more in PubMed

WHO Expert Committee on the Control of the Leishmaniases & World Health Organization. Control of the leishmaniases: report of a meeting of the WHO Expert Commitee on the Control of Leishmaniases, Geneva, 22-26 March 2010. Geneva: World Health Organization. http://www.who.int/iris/handle/10665/44412. Accessed 15 Mar 2018

Dantas-Torres F. Canine leishmaniosis in South America. Parasit Vectors. 2009;2(Suppl. 1):S1. doi: 10.1186/1756-3305-2-S1-S1. PubMed DOI PMC

Gállego M. Zoonosis emergentes por patógenos parásitos: las leishmaniosis. Rev Sci Tech. (OIE). 2004;23:661–676. doi: 10.20506/rst.23.2.1512. PubMed DOI

Franco AO, Davies CR, Mylne A, Dedet J-P, Gállego M, Ballart C, et al. Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables. Parasitology. 2011;138:1878–1891. doi: 10.1017/S003118201100148X. PubMed DOI

Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. Canine leishmaniosis - new concepts and insights on an expanding zoonosis: Part one. Trends Parasitol. 2008;24:325–330. doi: 10.1016/j.pt.2008.04.001. PubMed DOI

Borja LS, de Sousa OMF, Solcà MDS, Bastos LA, Bordoni M, Magalhães JT, et al. Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors. Vet Parasitol. 2016;229:110–117. doi: 10.1016/j.vetpar.2016.10.004. PubMed DOI

Molina R, Amela C, Nieto J, San-Andrés M, González F, Castillo JA, et al. Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Trans R Soc Trop Med Hyg. 1994;88:491–493. doi: 10.1016/0035-9203(94)90446-4. PubMed DOI

Alten B, Maia C, Afonso MO, Campino L, Jiménez M, González E, et al. Seasonal dynamics of phlebotomine sand fly species proven vectors of Mediterranean leishmaniasis caused by Leishmania infantum. PLoS Negl Trop Dis. 2016;10:e0004458. doi: 10.1371/journal.pntd.0004458. PubMed DOI PMC

Rioux JA, Guilvard E, Gállego J, Moreno G, Pratlong F, Portús M, et al. Intervention simultanée de Phlebotomus ariasi Tonnoir, 1921 et P. perniciosus Newstead, 1911 dans un même foyer. Infestations par deux zymodemes syntopiques. A propos d’une enquête en Catalogne (Espagne). In: Leishmania: Taxonomie et Phylogenèse Applications Éco-épidémiologiques. Montpellier: IMEEE; 1986. p. 439–44.

Guilvard E., Gallego M., Moreno G., Fisa R., Rispail P., Pratlong F., Martinez-Ortega E., Gallego J., Rioux J.A. Infestation naturelle dePhlebotomus ariasietPhlebotomus perniciosus(Diptera-Psychodidae) parLeishmania infantum(Kinetoplastida-Trypanosomatidae) en Catalogne (Espagne) Parasite. 1996;3(2):191–192. doi: 10.1051/parasite/1996032191. DOI

Ballart C, Guerrero I, Castells X, Barón S, Castillejo S, Alcover MM, et al. Importance of individual analysis of environmental and climatic factors affecting the density of Leishmania vectors living in the same geographical area: the example of Phlebotomus ariasi and P. perniciosus in northeast Spain. Geospat Health. 2014;8:389–403. doi: 10.4081/gh.2014.28. PubMed DOI

Aransay AM, Testa JM, Morillas-Marquez F, Lucientes J, Ready PD. Distribution of sandfly species in relation to canine leishmaniasis from the Ebro Valley to Valencia, northeastern Spain. Parasitol Res. 2004;94:416–420. doi: 10.1007/s00436-004-1231-4. PubMed DOI

Sáez VD, Morillas-Márquez F, Merino-Espinosa G, Corpas-López V, Morales-Yuste M, Pesson B, et al. Phlebotomus langeroni Nitzulescu (Diptera, Psychodidae) a new vector for Leishmania infantum in Europe. Parasitol Res. 2018;117:1105–1113. doi: 10.1007/s00436-018-5788-8. PubMed DOI

Rohousova I, Ozensoy S, Ozbel Y, Volf P. Detection of species-specific antibody response of humans and mice bitten by sand flies. Parasitology. 2005;130:493–499. doi: 10.1017/S003118200400681X. PubMed DOI

Vlkova M, Rohousova I, Drahota J, Stanneck D, Kruedewagen EM, Mencke N, et al. Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission. PLoS Negl Trop Dis. 2011;5:e1344. doi: 10.1371/journal.pntd.0001344. PubMed DOI PMC

Martín-Martín I, Molina R, Rohoušová I, Drahota J, Volf P, Jiménez M. High levels of anti-Phlebotomus perniciosus saliva antibodies in different vertebrate hosts from the re-emerging leishmaniosis focus in Madrid, Spain. Vet Parasitol. 2014;202:207–216. doi: 10.1016/j.vetpar.2014.02.045. PubMed DOI

Kostalova T, Lestinova T, Sumova P, Vlkova M, Rohousova I, Berriatua E, et al. Canine antibodies against salivary recombinant proteins of Phlebotomus perniciosus: a longitudinal study in an endemic focus of canine leishmaniasis. PLoS Negl Trop Dis. 2015;9:e0003855. doi: 10.1371/journal.pntd.0003855. PubMed DOI PMC

Marzouki S, Ben Ahmed M, Boussoffara T, Abdeladhim M, Ben Aleya-Bouafif N, Namane A, et al. Characterization of the antibody response to the saliva of Phlebotomus papatasi in people living in endemic areas of cutaneous leishmaniasis. Am J Trop Med Hyg. 2011;84:653–661. doi: 10.4269/ajtmh.2011.10-0598. PubMed DOI PMC

Carvalho AM, Cristal JR, Muniz AC, Carvalho LP, Gomes R, Miranda JC, et al. Interleukin 10-dominant immune response and increased risk of cutaneous leishmaniasis after natural exposure to Lutzomyia intermedia sand flies. J Infect Dis. 2015;212:157–165. doi: 10.1093/infdis/jiv020. PubMed DOI PMC

Gidwani K, Picado A, Rijal S, Singh SP, Roy L, Volfova V, et al. Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: a cluster-randomized trial. PLoS Negl Trop Dis. 2011;5:e1296. doi: 10.1371/journal.pntd.0001296. PubMed DOI PMC

Clements Meredith F., Kumar Vijay, Rijal Suman, Rogers Matthew E., Hostomska Jitka, Boelaert Marleen, Sundar Shyam, Gidwani Kamlesh, Hamilton Gordon, Das Pradeep, Picado Albert, Davies Clive R., Das Murari, Dinesh Diwakar S., Kumar Rajiv, Müller Ingrid, Volfova Vera, Volf Petr. Measurement of Recent Exposure to Phlebotomus argentipes, the Vector of Indian Visceral Leishmaniasis, by Using Human Antibody Responses to Sand Fly Saliva. The American Journal of Tropical Medicine and Hygiene. 2010;82(5):801–807. doi: 10.4269/ajtmh.2010.09-0336. PubMed DOI PMC

Volf P, Rohoušova I. Species-specific antigens in salivary glands of phlebotomine sandflies. Parasitology. 2001;122:37–41. doi: 10.1017/S0031182000007046. PubMed DOI

Lestinova T, Rohousova I, Sima M, de Oliveira CI, Volf P. Insights into the sand fly saliva: blood-feeding and immune interactions between sand flies, hosts, and Leishmania. PLoS Negl Trop Dis. 2017;11:e0005600. doi: 10.1371/journal.pntd.0005600. PubMed DOI PMC

Lestinova T, Vlkova M, Votypka J, Volf P, Rohousova I. Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate. Acta Trop. 2015;144:9–18. doi: 10.1016/j.actatropica.2015.01.005. PubMed DOI

Drahota J, Martin-Martin I, Sumova P, Rohousova I, Jimenez M, Molina R, et al. Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector. PLoS Negl Trop Dis. 2014;8:e2597. doi: 10.1371/journal.pntd.0002597. PubMed DOI PMC

Kostalova T, Lestinova T, Maia C, Sumova P, Vlkova M, Willen L, et al. The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis. Med Vet Entomol. 2016;31:88–93. doi: 10.1111/mve.12192. PubMed DOI

Hostomska J, Rohousova I, Volfova V, Stanneck D, Mencke N, Volf P. Kinetics of canine antibody response to saliva of the sand fly Lutzomyia longipalpis. Vector Borne Zoonotic Dis. 2008;8:443–450. doi: 10.1089/vbz.2007.0214. PubMed DOI

Sima M, Ferencova B, Warburg A, Rohousova I, Volf P. Recombinant salivary proteins of Phlebotomus orientalis are suitable antigens to measure exposure of domestic animals to sand fly bites. PLoS Negl Trop Dis. 2016;10:e0004553. doi: 10.1371/journal.pntd.0004553. PubMed DOI PMC

Rohousova I, Talmi-Frank D, Kostalova T, Polanska N, Lestinova T, Kassahun A, et al. Exposure to Leishmania spp. and sand flies in domestic animals in northwestern Ethiopia. Parasit Vectors. 2015;8:360. doi: 10.1186/s13071-015-0976-1. PubMed DOI PMC

Morillas Marquez F, Guevara Benitez DC, Ubeda Ontiveros JM, Gonzalez Castro J. Fluctuations annuelles des populations de Phlébotomes (Diptera, Phlebotomidae) dans la province de Grenade (Espagne) Ann Parasitol Hum Comp. 1983;58:625–632. doi: 10.1051/parasite/1983586625. PubMed DOI

Gálvez R., Descalzo M.A., Miró G., Jiménez M.I., Martín O., Dos Santos-Brandao F., Guerrero I., Cubero E., Molina R. Seasonal trends and spatial relations between environmental/meteorological factors and leishmaniosis sand fly vector abundances in Central Spain. Acta Tropica. 2010;115(1-2):95–102. doi: 10.1016/j.actatropica.2010.02.009. PubMed DOI

González E, Jiménez M, Hernández S, Martín-Martín I, Molina R. Phlebotomine sand fly survey in the focus of leishmaniasis in Madrid, Spain (2012–2014): seasonal dynamics, Leishmania infantum infection rates and blood meal preferences. Parasit Vectors. 2017;10:368. PubMed PMC

Lladró S, Picado A, Ballart C, Portús M, Gállego M. Management, prevention and treatment of canine leishmaniosis in north-eastern Spain: an online questionnaire-based survey in the province of Girona with special emphasis on new preventive methods (CaniLeish vaccine and domperidone) Vet Rec. 2017;180:47. doi: 10.1136/vr.103653. PubMed DOI

Quinnell RJ, Soremekun S, Bates PA, Rogers ME, Garcez LM, Courtenay O. Antibody response to sand fly saliva is a marker of transmission intensity but not disease progression in dogs naturally infected with Leishmania infantum. Parasit Vectors. 2018;11:7. doi: 10.1186/s13071-017-2587-5. PubMed DOI PMC

Marzouki S, Kammoun-Rebai W, Bettaieb J, Abdeladhim M, Hadj Kacem S, Abdelkader R. Validation of recombinant salivary protein PpSP32 as a suitable marker of human exposure to Phlebotomus papatasi, the vector of Leishmania major in Tunisia. PLoS Negl Trop Dis. 2015;9:e0003991. doi: 10.1371/journal.pntd.0003991. PubMed DOI PMC

Souza AP, Andrade BB, Aquino D, Entringer P, Miranda JC, Alcantara R, et al. Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral leishmaniasis endemic areas. PLoS Negl Trop Dis. 2010;4:e649. doi: 10.1371/journal.pntd.0000649. PubMed DOI PMC

Anderson JM, Oliveira F, Kamhawi S, Mans BJ, Reynoso D, Seitz AE, et al. Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genomics. 2006;7:52. doi: 10.1186/1471-2164-7-52. PubMed DOI PMC

Kedzierski L, Evans KJ. Immune responses during cutaneous and visceral leishmaniasis. Parasitology. 2014;141:1544–1562. doi: 10.1017/S003118201400095X. PubMed DOI

Andrade BB, Teixeira CR. Biomarkers for exposure to sand flies bites as tools to aid control of leishmaniasis. Front Immunol. 2012;3:121. doi: 10.3389/fimmu.2012.00121. PubMed DOI PMC

Kamhawi S. Protection Against Cutaneous Leishmaniasis Resulting from Bites of Uninfected Sand Flies. Science. 2000;290(5495):1351–1354. doi: 10.1126/science.290.5495.1351. PubMed DOI

Rohoušová I, Hostomská J, Vlková M, Kobets T, Lipoldová M, Volf P. The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure. Int J Parasitol. 2011;41:481–485. doi: 10.1016/j.ijpara.2011.01.003. PubMed DOI

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36:S1–S9. doi: 10.1111/j.1948-7134.2011.00106.x. PubMed DOI

Sanchez J, Dohoo IR, Markham F, Leslie K, Conboy G. Evaluation of the repeatability of a crude adult indirect Ostertagia ostertagi ELISA and methods of expressing test results. Vet Parasitol. 2002;109:75–90. doi: 10.1016/S0304-4017(02)00194-2. PubMed DOI

Ballart C, Alcover MM, Picado A, Nieto J, Castillejo S, Portús M, et al. First survey on canine leishmaniasis in a non classical area of the disease in Spain (Lleida, Catalonia) based on a veterinary questionnaire and a cross-sectional study. Prev Vet Med. 2013;109:116–127. doi: 10.1016/j.prevetmed.2012.09.003. PubMed DOI

Riera C, Valladares JE, Gállego M, Aisa MJ, Castillejo S, Fisa R, et al. Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate. Vet Parasitol. 1999;84:33–47. doi: 10.1016/S0304-4017(99)00084-9. PubMed DOI

Humoral and T-Cell-Mediated Immunity Against Phlebotomus perniciosus Salivary Proteins in Dogs from a Leishmaniosis-Endemic Area

Assessment of the exposure to Phlebotomus perniciosus and the presence of anti-Leishmania infantum antibodies in stray cats in an endemic region of Spain, and their potential correlation with environmental factors

Phlebotomus perniciosus Recombinant Salivary Proteins Polarize Murine Macrophages Toward the Anti-Inflammatory Phenotype

Monitoring Leishmania infection and exposure to Phlebotomus perniciosus using minimal and non-invasive canine samples

Exploring the relationship between susceptibility to canine leishmaniosis and anti-Phlebotomus perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in Mallorca, Spain

Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis

Antibody response to Phlebotomus perniciosus saliva in cats naturally exposed to phlebotomine sand flies is positively associated with Leishmania infection